Chromatin structure regulates gene expression patterns important for the maintenance of stem cell populations and for their differentiation into the various cell types of an organism. The local architecture of chromatin is influenced by chromatin modifying enzymes that alter the compaction state of nucleosomes to activate or silence genes. Polycomb group (PcG) proteins are chromatin modifiers that have central roles in the silencing of developmental genes that control the balance between differentiation and stem cell self-renewal. Defects in PcG protein function lead to developmental progression defects and are associated with an increased risk for cancer. One of the major questions in the field is how PcG proteins are recruited to sites of chromatin in mammalian genomes. Determining how PcG complexes are recruited is crucial for understanding the details of developmental regulation and may serve as a target to correct misregulation in aberrant cell types. There is an increasing association of long non-coding RNAs (lncRNAs) with the regulation of PcG protein function. Little is known about the functional role of lncRNAs in the cell and in particular how they impact PcG activity. We hypothesize that lncRNA interactions are necessary for recruitment of PcG proteins to specific silencing regions in order to influence developmental progression. The goal of this proposal is to determine the mechanistic details of the association between lncRNAs and PcG proteins on chromatin and to elucidate how these interactions affect polycomb group protein function. To achieve this goal, a high- throughput screen to discover the set of lncRNAs that interact with the PcG protein, PRC1 will be performed. Interacting lncRNAs will be identified by deep sequencing of RNA samples after immunoprecipitation of PRC1 complexes from human mesenchymal stem cells. A careful set of selection criteria will be administered to establish relevant lncRNA candidates and a thorough functional validation strategy will be used to discern biologically meaningful lncRNA interactions involved in gene silencing. In order to gain insight into the mechanism of action employed by lncRNAs on PRC1 function, the molecular determinants that govern the interaction between lncRNAs and PRC1 will be determined through a guided structure/function analysis. Together, these studies will provide a mechanistic basis for how lncRNAs interact with PcG complexes in order to silence developmental regulatory genes. Furthermore, uncovering mechanistic details of lncRNA-PcG interactions will establish a new framework for understanding epigenetic regulation and may ultimately provide a novel target for controlling cell fate determination and proliferation.